Production of Monoclonal Antibodies by Phage Display Technique

1. What is Phage Display Technology?

Phage display technology is a new technology that fuses foreign proteins or peptides with phage shell proteins, displays them on the phage surface and maintains a specific spatial conformation, and uses specific affinity to screen specific proteins or peptides.

2. Principle of Phage Display Technology

In 1985, Smith, G.P proposed to insert foreign genes into modified phage coat protein genes to express fusion proteins containing foreign proteins or peptides, and such phages with fusion proteins were called fusion phages. Through the repeated process of adsorption, elution and amplification, phages containing foreign proteins that can specifically bind to target proteins are selected from the phage library expressing various foreign proteins, and then enrichment, amplification and gene sequencing are performed to infer the amino acid composition of foreign proteins. This technique combines genotype and phenotype, molecular binding activity and phage extensibility, which is a new screening technique. At present, it has been successfully applied to antigen epitope analysis, monoclonal antibody screening, determination of protein function antagonistic polypeptides or simulated polypeptides, and has the advantages of large screening capacity, mass production by fermentation, and simple method. It plays an important role in vaccine design and so on.

3 Applications of Phage Libraries

Phage display antibody libraries are widely used to screen high-affinity antibodies, such as angiogenic marker fibronectin, tumor-specific B3/B4 antigen, EGFR and other protein-specific high-affinity antibodies. Antibody libraries constructed by phage display technology are usually derived from mRNAs derived from peripheral blood mononuclear cells, spleen cells, bone marrow cells and tonsils amplified by RT-PCR to form cDNA fragments. These gene fragments are splicted into phage vectors to form covalent structural proteins for expression and display in vitro. The heavy and light chains of antibodies are expressed independently and freely, and then randomly combined to form a huge antibody library with 10^11-10^12 phage clones.

4. Library Types

4.1 Fab Library

Fab fragment is the region of the antibody structure that can bind to the antigen, also known as the antigen-binding fragment, which involves the affinity of antibody-antigen binding, antibody specificity and other characteristics. The antibody Fab fragment contains 1 variable region (V region) and 1 constant region (C1), and the V region contains 3 complementary determination domains (CDR), which are involved in the production of unique type antibodies and the overfrequency mutation of antibodies. In particular, the CDR3 domain is the core region of antibody engineering. C1 zone mainly plays the role of structural support. Fab antibodies can be prepared by enzymolysis.

The construction of Fab antibody libraries is one of the effective ways to obtain high quality and high affinity drug antibodies. The high library capacity (10^9-10^12) of phage display Fab antibody libraries improves the speed of antibody engineering, such as the Production speed of recombinant human monoclonal antibodies and chimeric antibodies. The core of phage display Fab antibody library is to increase the number of independent phage display clones, and the variable region (VH and VL) genes of antibodies, especially the CDR region (complementary determination region), are formed by different strategies and methods, such as amino acid mutation, frameshift reading frame, etc. After sensitizing human peripheral blood mononuclear cells in vitro, mononuclear cells with high affinity were isolated by flow sorting technology and monoclonal treatment with EBV was carried out. By using this method, mononuclear cells with high consistency could be cloned, and then library construction was carried out.

4.2 scFv Library

Single-chain Fv (scFv) antibody library means that the variable region (VH and VL) genes of antibodies, especially the CDR region (complementary determining region), are formed into a large number of different clones by different strategies and methods, such as amino acid mutation, frameshift, etc. High affinity single chain Fv antibodies against the target antigen were screened from these clones. Studies have shown that the diversity of CDR3 in the variable region of antibodies directly determines the antigen specificity and binding affinity, and the diversity of CDR3-FR (frame region) combinations determines the affinity maturation process of antibodies.

The first level library with the capacity of 10^8-10^9CFU was constructed by using error-inclined PCR and nested PCR. Compared to Fab antibody libraries, scFV antibodies are more stable and less likely to form polymers due to their smaller molecular weight (about 25kDa).

4.3 VHH library

Alpacas are members of the camel family, which includes the Llama as well as the familiar Alpaca. In addition to regular IgG, camels also produce a functional single-chain antibody that is able to bind to an antigen, sometimes called a VHH antibody. These antibodies consist only of heavy chains, lacking light chains and functional CH1 and CH4 domains. These so-called heavy-chain antibodies are being used to develop single-domain antibodies. The single-domain nature of these antibodies provides unique advantages that make them stand out in biotechnology applications.

VHH antibody (nano antibody) surface display using pIII protein of M13 phage. Phage vectors were constructed, transfected into TG1 and XL1 Blue host cells, and after 2-3 transfections and amplification, high-quality VHH antibody display libraries with sufficient storage capacity of 10^8-10^9 and packaging capacity of 10^13-10^16/ml could be obtained. Up to 20-30 high-affinity phage strains could be obtained by screening multiple rounds of nanoantibody libraries by antigen-limiting pressure. Finally, through simple IPTG induction, a large number of antibody expression products can be obtained within 1-2 weeks.

KMD Bioscience has established a complete and mature phage antibody display technology platform. Based on the phage display technology platform, KMD Bioscience can provide major experimental links including antigen design, alpaca immunity, library construction and screening, and activity function verification, and provide highly specific and high-affinity alpaca VHH antibodies to scientists around the world. In addition, KMD Bioscience has rich experience in antibody engineering construction, and can provide three-dimensional antibody upstream and downstream services, including antibody humanization service, human scFv antibody library construction service, human Fab antibody library construction service, human antibody phage library Production service, phosphorylated antibody customization service, antibody affinity maturation service, etc.

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